Dot-ELISA法检测结核分枝杆菌特异性抗原CFP10的改进和建立

Development of a Dot-ELISA Method for Detecting Mycobacterium Tuberculosis-specific Antigen CFP10

目的建立一种检测结核分枝杆菌特异性抗原培养滤液蛋白10(culture filtrate protein 10,CFP10)的斑点酶联免疫吸附试验(dot enzyme linked i mmunesorbant assay,Dot-ELISA)方法。方法采用真空泵抽吸浓缩检测样本,改进Dot-ELISA用改进前的和改进后的Dot-ELISA方法检测同一批临床确诊的结核性胸膜炎的胸腔积液25例和非结核性胸腔积液31例,比较方法改进前后检测CFP10的敏感性和特异性;改变实验程序中的工作条件(硝酸纤维素膜的孔径、滴加样本的体积、真空泵抽吸的压力、不同的封闭液、温育的温度和时间及抗体的滴度),确定最适工作条件及重复性和稳定性。结果改进前检测CFP10的敏感性为76.0%,改进后并应用最适条件的敏感性为96.0%,差异有统计学意义(P=0.042);特异性和重复性、稳定性实验结果表明,所建立的Dot-ELISA法特异性和改进前差异无统计学意义(P=0.641),且稳定性和重复性较好。结论所建立的Dot-ELISA是一种比较敏感、特异和稳定的检测方法,能用于结核特异性抗原的检测和疾病的诊断。

Objective To develop a dot enzyme linked immunosorbent assay（Dot-ELISA）method for detecting culture filtrate protein 10（CFP10）.Methods Dot-ELISA method was improved using vacuum pump to concentrate test samples.Twenty-five cases of tuberculous pleural effusion and 31 cases of non-tuberculous pleural effusion from the same batch of cases were detected using the Dot-ELISA traditional method and the improved Dot-ELISA method,respectively,and the two methods were compared.The repeatability and stability,as well as the best working condition,were determined by changing the working condition,including aperture of nitrocellulose membrane,volume of samples,suction pressure,blocking buffer,temperature and time of incubation,and antibody titers.Results The sensibility of the traditional method and the improved method was 76.0% and 96.0%,respectively,and there was a significant difference between the two groups（P=0.042）.But the specificity of the traditional method was not statistically different from the improved method（P=0.641）.The repeatability and stability of the improved method were better than those of the traditional method.Conclusion The developed Dot-ELISA method is sensitive,specific and stable,and can be used for detection of mycobacterium tuberculosis-specific antigen and diagnosis of tuberculosis.